The heavy and light chains of RoAb13 were amplified from the hybridoma cDNA, by rapid amplification of cDNA ends (5’ RACE). Briefly, the RNA from the hybridoma was reverse transcribed using constant region primers. The 5’ end of the transcript was extended by addition of adenosines using terminal deoxynucleotidyltransferase, and the resulting molecules were amplified using a poly T primer and 3’ constant region primers ([22 (link)][23 (link)]. The primers used are given in Supporting Information S1 Table . The PCR amplicons were cloned into T-vector (Life Technologies) and sequenced by Sanger sequencing at the UCL Genomics facility. The sequences have been submitted to EMBL with accession numbers LN832627 and LN832628.
T vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The T-vector is a plasmid DNA vector designed for the cloning of PCR products. It features a single 3' terminal thymidine (T) residue that allows for the direct insertion of PCR products amplified with DNA polymerases that add a single adenosine (A) residue to the 3' ends of the amplified fragments. The T-vector facilitates the rapid cloning of PCR products without the need for additional enzymatic modifications.
Automatically generated - may contain errors
Lab products found in correlation
Universal reverse primer, by Qiagen (1 mentions)
Miscript sybr green, by Qiagen (1 mentions)
Thermoscript rt, by Thermo Fisher Scientific (1 mentions)
Miscript 2 rt kit, by Qiagen (1 mentions)
Mirneasy, by Qiagen (1 mentions)
Gotaq green, by Promega (1 mentions)
Taq polymerase, by Thermo Fisher Scientific (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Proteinase k, by Avantor (1 mentions)
Ez dna methylation directtm kit, by Zymo Research (1 mentions)
Firstchoice rlm 5 race kit, by Thermo Fisher Scientific (1 mentions)
Abi 3700 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Top10, by Thermo Fisher Scientific (1 mentions)
Pepstatin a, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Bestatin, by Merck Group (1 mentions)
8 protocols using t vector
1
Cloning and Sequencing of RoAb13 Antibody
2
Total RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from cells using miRNeasy (Qiagen, Valencia, CA) as previously described [57 (link)]. Equal amounts of RNA were reverse transcribed into cDNA using the miScript II RT Kit and qRT-PCR was conducted with miScript SYBR Green (Qiagen). mRNA expression was normalized to beta actin, while small RNA expression was normalized to RNU6B (RNU6B_13, Qiagen). The polyA site was identified in BCAR3 from PEO4 and 2008 cells using Thermoscript RT (Invitrogen) and oligo dT as previously described [58 ]. PCR products were amplified using the 5’ forward BCAR3 primer with the Universal reverse primer from Qiagen and amplified using GoTaq Green (Promega, Madison, WI). Primers are listed in Supplementary Table 3 . PCR products were cloned into T-vector (Life Technologies, Carlsbad, CA) and sent for direct sequencing (University of Minnesota Genomic Center, Minneapolis, MN).
3
Bisulfite Sequencing and Methylation-Specific PCR
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Primers used in the MSP and BSP were listed in Supplementary Table S5 . The methylation status of the target genes in the twins and CEM*174 cell lines were determined by bisulfite sequencing using EZ DNA Methylation-Gold™ Kit (Zymo Research) as instructed. The target regions were amplified using Taq polymerase (Invitrogen) cloned into T-vector (Invitrogen) and sequenced as instructions. The resulting sequences of the bisulfite treated DNA were aligned with the reference genome and reported in the form of “lollipop” diagram by BiQ Analyzer54 (link). Methylation specific PCR was performed to analyze the methylation status of the target genes in the HIV/AIDS patients and normal subjects. Standard Human WGA Non-Methylated DNA (Zymo Research) were used as positive control in PCR for unmethylated CpGs and negative control in PCR for methylated CpGs; while conversely, Standard Human WGA Methylated (Zymo Research) were used as negative control in PCR for unmethylated CpGs and positive control in PCR for methylated CpGs
4
Quantifying Oocyte mtDNA Methylation
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A total of approximately 120 oocytes per group were used for mtDNA methylation analysis. Oocytes were incubated with lysis solution (0.5 mol/L EDTA, pH 8.0; 2 mg/mL proteinase K, Amresco, USA) for 50 min at 37 °C and followed by bisulfite treatment using the EZ DNA Methylation-DirectTM Kit (Zymo Research, USA) according to the manufacturer’s instructions. Modified DNA was then used as a template in PCR amplification with the primers listed in Table S1 . The PCR products were cloned to T vector and sequenced (Invitrogen, USA).
5
Determination of HBL1 Transcript Sequence
6
Designing Primers for Crab HSP90 Gene
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The specific primers of C. japonica HSP90 gene designed using consensus comparison of Brachyura sequences. Multiple sequence alignments were conducted using ClustalW in Mega 4.0. Specific primers of C. japonica HSP90 were designed from orthologue sequences of Chinese mitten crab (Eriocheir sinensis, GenBank accession No. ADE60732), swimming crab (Portunus trituberculatus, GenBank accession No. ACQ90225) and green mud crab (Scylla paramamosain GenBank accession No. ACN54679). Information of primer sequences was shown in Table 1 . The size of polymerase chain reaction (PCR) products were 442 bp for the HSP90 gene and 233 bp for the beta-actin gene. The condition of PCR mixture with a total volume of 50 µL contained 1×Taq polymerase buffer, 200 µM dNTP, 5 units of Taq polymerase, primers at concentrations of 10 µM and 0.5 M betaine. The PCR conditions were finally optimized as this protocol: one cycle of 94℃ for 5 minutes, 40 cycles of 94℃ for 30 seconds, 55℃ for 40 seconds, 72℃ for 1 minute and one cycle of 72℃ for 7 minutes. The amplified PCR products were cloned in the T-vector (Invitrogen, Inc., Carlsbad, CA, USA) and sequencing with an ABI 3700 genetic analyzer (Applied Biosystems, Foster City, CA, USA).
7
Salmonella Serotypes and E. coli Expression
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The three Salmonella serotypes, Salmonella Typhimurium, Salmonella Enteritidis and Salmonella Gallinarum (Table 1 ), were obtained from Dr. Jin Hur (Chonbuk National University, Iksan, Korea). The synthetic MP-V1 used in the previous study [25 (link)] was also used in this study. The protease inhibitor cocktail for bacterial use and the protease inhibitors, including AEBSF, bestatin, pepstatin A, E-64 and EDTA, were purchased from Sigma-Aldrich (Milwaukee, WI, USA). An E. coli strain and plasmids, used for the construction of the E. coli secretion system, are listed in Table 1 , and Top10 (an E. coli competent cell) and the T-vector were purchased from Invitrogen (Carlsbad, CA, USA) and Promega (Madison, WI, USA), respectively.
8
Cloning and Sequencing of ssDNA Aptamers
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The ssDNA aptamers pool of 9 rounds was amplified by PCR to obtain double-stranded DNAs (dsDNAs) using unmodified primers and then cloned into a T vector (Invitrogen). The recombinant plasmid was then transformed into E. coli DH5α and randomly selected 50 clones using blue-white selection. The selected 50 clones were sequenced by Invitrogen Co., Ltd. (Shanghai, China) and designated as LY1 to LY50.
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